applied sciences

Article Isolation and Optimization of ruber OMNRC45 for Red Pigment Production and Evaluation of the Pigment as a Food Colorant

Osama M. Darwesh 1, Ibrahim A. Matter 1,* , Hesham S. Almoallim 2, Sulaiman A. Alharbi 3 and You-Kwan Oh 4,*

1 Department of Agricultural Microbiology, National Research Centre, 33 El-Bohouth St., Dokki, Cairo 12622, Egypt; [email protected] 2 Department of Oral and Maxillofacial Surgery, College of Dentistry, King Saud University, Riyadh 11461, Saudi Arabia; [email protected] 3 Department of Botany & Microbiology, College of Science, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] 4 Department of Chemical & Biomolecular Engineering, Pusan National University, Busan 46241, Korea * Correspondence: [email protected] (I.A.M.); [email protected] (Y.-K.O.); Tel.: +20-1155244667 (I.A.M.); +82-51-510-2395 (Y.-K.O.)  Received: 20 November 2020; Accepted: 9 December 2020; Published: 11 December 2020 

Abstract: The color of food is a critical factor influencing its general acceptance. Owing to the effects of chemical colorants on health, current research is directly aimed at producing natural and healthy food colorants from microbial sources. A pigment-producing , obtained from soil samples and selected based on its rapidity and efficiency in producing red pigments, was identified as Monascus ruber OMNRC45. The culture conditions were optimized to enhance pigment production under submerged fermentation. The optimal temperature and pH for the highest red pigment yield were 30 ◦C and 6.5, respectively. The optimum carbon and nitrogen sources were rice and peptone, respectively. The usefulness of the pigment produced as a food colorant was evaluated by testing for contamination by the harmful mycotoxin citrinin and assessing its biosafety in mice. In addition, sensory evaluation tests were performed to evaluate the overall acceptance of the pigment as a food colorant. The results showed that M. ruber OMNRC45 was able to rapidly and effectively produce dense natural red pigment under the conditions of submerged fermentation without citrinin production. The findings of the sensory and biosafety assessments indicated the biosafety and applicability of the red Monascus pigment as a food colorant.

Keywords: Monascus ruber; food colorant; citrinin mycotoxin; biosafety evaluation

1. Introduction In addition to taste, aroma, and texture, the color of food is a main factor that influences its total acceptability. Therefore, since ancient times, people have used multiple colorants in their food to improve its appearance, flavor, taste, preservability, and nutrient quality. Food colorants can be obtained either from natural sources (plants, animals, or ) or through chemical synthesis [1,2]. Owing to the differences in the origin and composition of the applied food colorants, the degree of health risk and environmental suitability varies widely [3,4]. Chemically synthesized food colorants are not preferred because of their potential toxicity, carcinogenicity, and undesirable side effects on human health and the environment. Natural food pigments are gaining increasing

Appl. Sci. 2020, 10, 8867; doi:10.3390/app10248867 www.mdpi.com/journal/applsci Appl. Sci. 2020, 10, 8867 2 of 15 popularity globally, as they are safe to use and have several nutritional benefits and potential medicinal properties [4–6]. Microorganisms (bacteria, fungi, and microalgae) are among the most promising natural sources of food colorants, which can be produced both intracellularly and extracellularly. They are characterized by high, sustainable productivity and relatively low costs, as they can be cultured on agricultural and food wastes at any time of the year. In addition, the pigments obtained are varied, easily extractable, and relatively heat-stable, offering several health and nutritional benefits [7,8]. The Monascus was widely used in East Asian countries approximately two thousand years ago, and it continues to be used for the fermentation and coloring of foods, particularly rice (to produce red rice), which is also referred to as “red rice”, “hongqu,”, “red rice”, “red koji”, and “anka” in traditional medicine. However, the name “Monascus spp.” was proposed only in 1884 for a versatile group of filamentous fungi comprising Monascus floridanus, Monascus pallens, Monascus pilosus, , Monascus ruber, , Monascus eremophilus, Monascus lunisporas, and Monascus argentinensis [9–11]. Pigments produced by Monascus by natural fermentation have a high economic value and have attracted considerable attention as coloring agents globally. They have several advantages, such as ease of production using inexpensive substrates, favorable solubility in acidic water and ethanol, presence of numerous bioactive metabolites, and safety when produced under specific conditions [12]. Monascus spp. are able to synthesize different (intracellular and extracellular) polyketide secondary metabolites that protect cells against oxidative stress (antioxidants) and produce different and distinct pigments. The most common pigments produced by Monascus are orange (monascorubrin and rubropunctatin), yellow (monascin and ankaflavin), and hydrosoluble red (monascorubramine and rubropunctamine). The percentage of production of each pigment by Monascus is highly dependent on the species and cultivation conditions (substrate, fermentation mode, dissolved oxygen, pH, and temperature). Of the several pigments produced by Monascus, the red pigment (monascorubramine) is in high demand [4,13–15]. Solid-state fermentation of rice and grains is the traditional method for cultivating Monascus to produce high levels of pigments, although it has been linked to scale-up- and contamination-related issues [16]. In addition, traditional solid-state fermentation processes are labor-intensive, time-consuming, and require large cultivation areas [17,18]. Monascus strains usually produce lower levels of pigment in submerged cultivation than in solid-state cultivation [19]. However, the production of pigments by Monascus under submerged fermentation is characterized by controlled cultivation conditions and medium, which helps to control the production of secondary metabolites [10]. During the growth of certain Monascus strains, the mycotoxin citrinin is produced as a secondary metabolite under specific fermentation conditions [20,21]. Citrinin is nephrotoxic, hepatotoxic, and carcinogenic to various animals and humans; therefore, its presence in food products is a critical issue. Previous studies have shown that the citrinin production gene is only harbored by M. purpureus and M. kaoliang (M. kaoliang is a synonym for M. purpureus) and not by M. pilosus, M. ruber, M. floridanus, M. sanguineus, M. barkerior, or M. lunisporas. However, M. ruber has been reported to produce citrinin under certain fermentation conditions [10]. In addition, an “uncommon case of M. ruber invasive gastric infection associated with the consumption of contaminated dried and salted fish” has been reported [22]. In this study, we isolated a fungus that could efficiently produce safe red pigments for use as food colorants, with an emphasis on preventing the production of mycotoxins (e.g., citrinin) as secondary metabolites. Additionally, we attempted to improve pigment production by controlling culture conditions, and the pigment produced was extracted and tested as a food colorant in specific food products for children (jellybeans and lollipops). Appl. Sci. 2020, 10, 8867 3 of 15

2. Materials and Methods

2.1. Samples and Isolation Sources Nine different samples were collected from various sites in Egypt exposed to spillage of intact or broken rice grains in addition to rice processing residues (wastes). Among the samples, four soil samples were collected from places near rice storage and hulling machines, while two soil samples were collected from rice paddy fields (Kafr El-Sheikh Governorate). In addition, the remaining three samples were collected from wastewater of a local food processing market (in Giza Governorate). The samples were collected in sterilized jars and transferred to the laboratory under aseptic conditions, as previously described by Darwesh et al. [23].

2.2. Isolation of Red Pigment-Producing Fungi The samples were enriched by culturing with 10% (w/v) sterilized broken rice (1–2 mm diameter) to increase the chances of isolating red pigment-producing fungi. The pigment-producing fungi were isolated on solidified rice agar, which was prepared as described by Wu et al. [16] with certain modifications: the medium contained 15 g/L rice powder, 0.5 g/L KH2PO4, 1.0 g/LK2HPO4, 0.1 g/L NaCl, 0.2 g/L MgSO 7H O, and 1.0 g/L NH NO , and the pH was adjusted to 5.5–6. After 7 days of culture on 4· 2 4 3 rice agar plates at 28 2 C, the fungal colonies that developed and produced red pigments were selected ± ◦ and re-cultured on potato dextrose agar (PDA) for purification and preservation. The selected fungal isolates were screened to assess the intense and rapid production of red pigments (morphologically). For screening, individual fungal discs (9.5 mm in diameter) were cut from PDA Petri dishes and placed at the center of the surface of rice agar plates and incubated for 7 days at 28 2 C. For evaluating the ± ◦ production of red pigments, the fungal discs that produced the most intense and widely dispersed pigments rapidly were selected for subsequent studies.

2.3. Identification of the Target Fungal Strain The selected fungal isolate that exhibited satisfactory red pigment production was identified and characterized morphologically as well as by using molecular biology techniques. The morphological characteristics were evaluated using a light microscope (model cx41, Olympus, Tokyo, Japan) after 3 days of culturing on PDA plates [24]. Molecular identification of the fungal isolate was performed after culturing for 3 days on potato dextrose broth (PDB) and harvesting using a filter paper (Whatman No. 1, Sigma-Aldrich). Total genomic DNA was extracted using the CTAB protocol after drying and grinding of the mycelium using liquid nitrogen [25]. DNA from the fungal isolate was amplified by polymerase chain reaction (PCR) using internal transcribed spacer 1 (ITS1) (50-TCCGTAGGTGAACCTGCGG-30) and ITS4 (50-TCCTCCGCTTATTGATATGC-30) primers designed for sequencing. The sequence was identified by comparing the contiguous DNA sequence with data from the reference and type strains available in GenBank using the BLAST program (National Centre for Biotechnology Information). The obtained sequences were aligned using the Jukes Cantor Model [26].

2.4. Optimization of Extracellular Red Pigment Production Various environmental and nutritional parameters, such as temperature, pH, and carbon and nitrogen sources, were evaluated to improve and optimize the production of red pigments from the fungal strain under study. The optimization experiments were performed in 250 mL conical flasks containing 100 mL of modified rice broth medium (submerged fermentation), with incubation for 7 days (unless otherwise indicated) in an orbital incubator shaker. Red pigment production was indirectly evaluated by measuring the absorbance of the culture filtrate at 500 nm using a spectrophotometer (after centrifugation at 4000 rpm for 5 min) [27,28]. Instead of using the carbon or nitrogen sources present in the original medium, different components were added to the fermentation medium (rice broth medium) as carbon and nitrogen sources. The carbon sources included rice powder, wheat flour, corn flour, corn cob powder, and glucose Appl. Sci. 2020, 10, 8867 4 of 15

at a concentration of 10 g/L, whereas the nitrogen sources included (NH4)2SO4, NH4NO3, KNO3, peptone, and urea (quantities were calculated to be equivalent to 100 mg/L). As important environmental parameters, the incubation temperature (20, 25, 28, 30, 35, and 40 ◦C) and initial pH values (4.5, 5.5, 6.0, 6.5, 7.5, and 8.5) in the medium were modulated to study their effect on the production of red pigments by the fungal strain. In addition, another experiment was performed to evaluate the production of red dyes under different incubation periods (4, 6, 8, 10, 12, 14, 16, 18, and 20 days) at the optimum temperature and pH with the highest pigment yield.

2.5. Extraction of Pigments and Secondary Metabolites The optimized carbon and nitrogen sources from the previous experiment were used for fermentation of the selected fungal strain under submerged conditions to study the secondary metabolites produced. Static submerged fermentation was initiated by inoculating five activated fungal agar discs in a 500 mL flask containing 350 mL of modified liquid rice medium (pH 6.0). After incubation for 5 days at 28 2 C, the mycelia were harvested by filtration and washed twice ± ◦ with distilled water. The mycelium-free supernatant and wash mixture were collected and centrifuged at 4000 rpm for 5 min (to remove impurities) after volume adjustment for quantitative calculation. The washed mycelia were extracted by vigorous stirring for 12 h in 200 mL of 95% ethanol followed by centrifugation. Both aqueous and ethanolic extracts were subjected to spectrophotometric analysis at 500 nm for quantifying the red pigments produced.

2.6. Citrinin Analysis The presence of citrinin in the rice products fermented using the selected fungal strain was analyzed in 10-day-old cultures containing broken rice, under solid or liquid fermentation conditions. Citrinin extraction was performed using 100 mL of chloroform per 50 g of culture-mycelium mixture after homogenization in a high-speed mixer (Overhead Stirrer NOHS-100, Labnics, London, UK) at 16,000 rpm for 5 min. The extraction process was repeated three times, following which the chloroform extracts were combined, washed, filtered, and concentrated until near dryness. Next, thin-layer chromatography (TLC) was performed to detect citrinin [29].

2.7. Biosafety Evaluation The biological toxicity of the produced red pigment was evaluated in mice (according to the appropriate and recognized ethics) as follows: Young adult male mice (white mice, Mus musculus domesticus) weighing 25–30 g were obtained from the animal house at King Saud University, Riyadh, Saudi Arabia. The animals were housed in polycarbonate boxes at room atmosphere in the laboratory and acclimated for 1 week before the experiments commenced. The mice were divided into 13 groups, and the control and treatment groups comprised six mice each. The animals were fed a basal diet containing 20% casein, 10% sugar cane, 50% corn starch, 10% corn oil, 1% vitamin mixture, 5% cellulose, and 4% salt mixture [30]. The animals were provided with free access to water. Next, two types of pigments—natural (fungal) and synthetic red pigments (carmoisine E122; Sigma-Aldrich, India) —were fed by oral dosing for comparison. An acute toxicity test was performed to determine the oral lethal dose (LD50) of the two different colorants separately. The male white mice were randomly divided into natural- and carmoisine-fed groups. Each group was divided into six subgroups consisting of six animals that received six different doses of each pigment orally (10, 100, 1000, 1600, 2900, and 5000 mg/kg body weight). The mice in the control group received distilled water instead of pigments. The animals were observed thoroughly for the onset of any immediate toxic signs as well as during the observation period of 1 week. Motor activity, tremors, convulsions, aggressiveness, sedation, muscle relaxation, hypnosis, analgesia, ptosis, lacrimation, paralysis, diarrhea, and skin color were evaluated in the first 1 h, 24 h, and 1 week of pigment administration. The death rate was also recorded in each group. For hematological and serum biological analyses, at the end of the observation period, blood samples were collected and centrifuged to separate the serum. The levels of aspartate aminotransferase Appl. Sci. 2020, 10, 8867 5 of 15

(AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and blood urea nitrogen (BUN) were measured in the serum samples using kits from Biomed Diagnostics (Egypt). At the end of the experimentation, the animals used for blood sampling were sacrificed and autopsied. The liver, heart, spleen, and kidney were harvested, and the weight of each organ, along with the total body weight, was measured. The organs were also observed for major changes in appearance before weighing. The weight of each organ relative to the total body weight was calculated. The hepatosomatic index (HIS) was calculated as the percentage of liver weight upon body weight [31].

2.8. Application of the Obtained Red Pigment as a Food Colorant As sample food products, lollipops were prepared by mixing sucrose (242.4 g), corn syrup (129.5 mL), water (126.6 mL), and citric acid (0.75 g) and heating the mixture to 157 ◦C. The mixture was rapidly cooled to 110 ◦C, and 1.05 g of a flavoring agent as well as the natural fungal red pigment was added to produce red-colored lollipops. Jellybeans were prepared by mixing the ingredients using the traditional procedure (15 g of gelatin mixed with 85 g of purified sucrose, 0.2% citric acid, flavoring agent, and a mixture of sodium benzoate and potassium citrate at 0.1%). The concentrated fungal red pigment (0.1%) was added after mixing the ingredients, following which 0.1% (w/w) of ascorbic acid was added to the jelly. The jellies were placed in in a refrigerator until they solidified and then were packed in a special foil. The control was prepared using 0.1% carmoisine E122. Sensory evaluation was performed by ten panelists. The panelists were asked to evaluate the taste, color, texture, odor, and overall acceptability of the prepared food products.

2.9. Statistical Analyses The experiments were performed in triplicate, and the results are expressed as the mean standard ± deviation (SD). Statistical significance was evaluated using analysis of variance (SAS Studio 3.8, SAS Institute Inc., Cary, NC, USA) followed by the determination of the least significant difference (LSD) at 0.05.

3. Results and Discussion

3.1. Isolation and Identification of Pigment-Producing Fungal Strains Owing to the hazards associated with chemical colorants, in the current study, we attempted to directly produce natural and environmentally safe food colorants from fungal sources. To this end, nine samples suspected to contain pigment-producing fungi (based on their history of exposure to rice residues) were used as isolation sources of the target fungi. Six soil samples and three wastewater samples collected from food processing facilities were enriched using broken rice as the preferred carbon source to isolate pigment-producing fungi. Following the initial isolation process, twenty pigment-producing fungal isolates were obtained (Figure1), which were subsequently screened to select the most efficient strain in terms of pigment production. The isolated fungi were screened for their ability to produce pigments on rice agar medium plates. The rice medium used in this experiment was selected based on previous reports of its use as a preferred carbon source for multiple edible/pigment-producing fungi [6,32]. Pigmentation of various colors (red, yellow, orange, and violet) was observed on the incubated rice agar medium plates (Figure1). However, the red pigment was observed to be the most highly dispersible pigment, which was produced by fungal isolate No. 2 (isolated from soil samples enriched with broken rice). For confirmation, the fungal isolates were re-cultured in rice broth medium to confirm the production of pigments. It was clearly observed in both broth and solid cultures that fungal isolate No. 2 could rapidly produce an intense red pigment (Figure2). The produced pigment showed an absorption maximum at a wavelength of 500 nm, as shown in Figure2. Accordingly, fungal isolate No. 2 was selected for the subsequent studies. Appl. Sci. 2020, 10, x FOR PEER REVIEW 5 of 15

2.8. Application of the Obtained Red Pigment as a Food Colorant As sample food products, lollipops were prepared by mixing sucrose (242.4 g), corn syrup (129.5 mL), water (126.6 mL), and citric acid (0.75 g) and heating the mixture to 157 °C. The mixture was rapidly cooled to 110 °C, and 1.05 g of a flavoring agent as well as the natural fungal red pigment was added to produce red-colored lollipops. Jellybeans were prepared by mixing the ingredients using the traditional procedure (15 g of gelatin mixed with 85 g of purified sucrose, 0.2% citric acid, flavoring agent, and a mixture of sodium benzoate and potassium citrate at 0.1%). The concentrated fungal red pigment (0.1%) was added after mixing the ingredients, following which 0.1% (w/w) of ascorbic acid was added to the jelly. The jellies were placed in molds in a refrigerator until they solidified and then were packed in a special foil. The control was prepared using 0.1% carmoisine E122. Sensory evaluation was performed by ten panelists. The panelists were asked to evaluate the taste, color, texture, odor, and overall acceptability of the prepared food products.

2.9. Statistical Analyses The experiments were performed in triplicate, and the results are expressed as the mean ± standard deviation (SD). Statistical significance was evaluated using analysis of variance (SAS Studio 3.8, SAS Institute Inc., Cary, NC, USA) followed by the determination of the least significant difference (LSD) at 0.05.

3. Results and Discussion

3.1. Isolation and Identification of Pigment-Producing Fungal Strains Owing to the hazards associated with chemical colorants, in the current study, we attempted to directly produce natural and environmentally safe food colorants from fungal sources. To this end, nine samples suspected to contain pigment-producing fungi (based on their history of exposure to rice residues) were used as isolation sources of the target fungi. Six soil samples and three wastewater samples collected from food processing facilities were enriched using broken rice as the preferred carbon source to isolate pigment-producing fungi. Following the initial isolation process, twenty Appl. Sci. 2020, 10, x FOR PEER REVIEW 6 of 15 pigment-producingAppl. Sci. 2020, 10 ,fungal 8867 isolates were obtained (Figure 1), which were subsequently6 ofscreened 15 to select theThe most isolated efficient fungi strain were in screened terms of for pigment their abil production.ity to produce pigments on rice agar medium plates. The rice medium used in this experiment was selected based on previous reports of its use as a preferred carbon source for multiple edible/pigment-producing fungi [6,32]. Pigmentation of various colors (red, yellow, orange, and violet) was observed on the incubated rice agar medium plates (Figure 1). However, the red pigment was observed to be the most highly dispersible pigment, which was produced by fungal isolate No. 2 (isolated from soil samples enriched with broken rice). For confirmation, the fungal isolates were re-cultured in rice broth medium to confirm the production of pigments. It was clearly observed in both broth and solid cultures that fungal isolate No. 2 could rapidly produce an intense red pigment (Figure 2). The produced pigment showed an absorption maximum at a wavelength of 500 nm, as shown in Figure 2. Accordingly, fungal isolate No. 2 was selected for the subsequent studies. The fungal isolate was characterized and identified based on its morphological, culture, and molecular biology properties. In the microscopic observation of the fungal isolate, the asexual form with a chain of conidia and the sexual form with thin-walled ascoscarps containing oval ascospores were observed. These characteristics, in addition to the culture characteristics, indicated that the obtained isolate was of M. ruber [33]. Moreover, molecular biology techniques were applied to validate the results. Genomic DNA was isolated and purified according to a previously described protocol using isopropyl alcohol [34,35]. The ITS genes were sequenced after amplification using PCR. The obtained sequences were compared with related sequences available in GenBank using BLAST. The phylogenetic tree of the fungal isolate is shown in Figure 2, where its sequence was observed to show 99.45% similarity with the reference strain M. ruber in GenBank. Accordingly, it was named Monascus ruber OMNRC45 and registered and deposited under the same name with the FigureFigure 1. Enrichment 1. Enrichment and and purification purification of of pigment-producing pigment-producing fungi. fungi. National Research Centre (NRC), Egypt.

FigureFigure 2. Red 2. Redpigment-producing pigment-producing fungal fungal isolate isolate (N (No.o. 2) 2)with with the the highest highest potency: potency: ( a) its growthgrowth on rice agaron rice medium, agar medium, (b) pigment (b) pigment produced produced in inliquid liquid medium, medium, ( (cc) wavelengthwavelength scanning scanning using using a UV a UV spectrophotometer,spectrophotometer, and and (d) ( dphylogenetic) phylogenetic tree tree identification identification as asMonascus Monascus ruber ruberOMNRC45. OMNRC45.

3.2. OptimizationThe fungal of Red isolate Pigment was Production characterized by M. and ruber identified OMNRC45 based on its morphological, culture, and molecular biology properties. In the microscopic observation of the fungal isolate, the asexual form Pigmentwith a chain productivity of conidia and can the be sexual influenced form with by thin-walledthe microbial ascoscarps strain, containing medium ovalcomposition, ascospores and fermentation conditions [36]. Hence, to maximize the yield of red pigments produced by M. ruber OMNRC45, the environmental and culture conditions were improved. Carbon is an essential nutrient for the biosynthesis of cellular components, such as carbohydrates, proteins, and fats, and through oxidation, it provides the energy necessary for cells. For Monascus spp., carbon plays a critical role in cell growth, metabolism, and pigment production [37,38]. In the present study, different carbon Appl. Sci. 2020, 10, 8867 7 of 15 were observed. These characteristics, in addition to the culture characteristics, indicated that the obtained isolate was of M. ruber [33]. Moreover, molecular biology techniques were applied to validate the results. Genomic DNA was isolated and purified according to a previously described protocol using isopropyl alcohol [34,35]. The ITS genes were sequenced after amplification using PCR. The obtained sequences were compared with related sequences available in GenBank using BLAST. The phylogenetic tree of the fungal isolate is shown in Figure2, where its sequence was observed to show 99.45% similarityAppl. Sci. 2020 with, 10 the, x FOR reference PEER REVIEW strain M. ruber in GenBank. Accordingly, it was named Monascus7 of 15 ruber OMNRC45 and registered and deposited under the same name with the National Research Centre (NRC),sources Egypt. (i.e., rice powder, wheat flour, corn flour, corn cob powder, and glucose) were used to improve the growth of M. ruber OMNRC45 (in liquid medium) and its red pigment yield. The results 3.2.presented Optimization in Figure of Red 3a Pigmentshow that Production under the experimental by M. ruber OMNRC45conditions, rice powder was the best source of carbon for biomass and red pigment production. These results were consistent with the findings reportedPigment by Carvalho productivity et al. can[29], who be influencedobtained theby highest the pigment microbial yield strain, using mediumrice powder composition, as a andcarbon fermentation source. Through conditions the results [36]. Hence, obtained, to the maximize higher yield the yieldof red of pigments red pigments in the case produced of using by riceM. ruber OMNRC45,flour as a carbon the environmental source can be linked and culture to its higher conditions content were of starch improved. compared Carbon to other is carbon an essential sources nutrient forused. the biosynthesis This is supported of cellular by the results components, obtained suchby Lo asng carbohydrates,et al. [39], who indicated proteins, that and the fats, increase and in through oxidation,the production it provides of Monascus the energy pigments necessary is directly for related cells. to For the Monascusefficiency inspp., α-amylase carbon production plays a critical and role in cellstarch growth, degradation metabolism, by M. ruber and. Conversely, pigment productioncrushed corn [cob37, 38did]. not In lead the presentto high growth study, or di pigmentfferent carbon sourcesyield, (i.e.,which rice could powder, be attributed wheat flour,to the cornneed flour,for additional corn cob pre-treatment powder, and when glucose) using were this usedsubstance to improve to match the growth requirements of the fungal strain. the growth of M. ruber OMNRC45 (in liquid medium) and its red pigment yield. The results presented For similar reasons, the source of nitrogen in the culture medium is an essential factor in Figureinfluencing3a show microbial that under growth the as experimental well as the prod conditions,uction of rice primary powder and/ wasor secondary the best source bioactive of carbon formetabolites biomass and [40]. red As pigment secondary production. metabolites, These the pr resultsoduction were of consistentboth intracellular with the and findings extracellular reported by Carvalhopigments et is al. expected [29], who to be obtained influenced the (quantitativel highest pigmenty and qualitatively) yield using by rice the powder nitrogen assource a carbon used. source. ThroughWe investigated the results the obtained, effects of theorganic higher (peptone–urea) yield of red and pigments inorganic in (NH the4 caseNO3-(NH of using4)2SO4 rice-KNO flour3) as a carbonnitrogen source sources can on be the linked growth to itsof M. higher ruber contentOMNRC45 of starchand extracellular compared red to pigment other carbon productivity. sources used. ThisAs is shown supported in Figure by the 3b, resultsM. ruber obtained OMNRC45 by Longcultured et al.using [39 ],peptone who indicated as the nitrogen that the source increase (in in the productioncultivation of medium)Monascus showedpigments the highest is directly pigment related yield. to However, the efficiency the increase in α-amylase in fungal production biomass and production using the same nitrogen source was not significant compared to the results obtained using starch degradation by M. ruber. Conversely, crushed corn cob did not lead to high growth or pigment ammonium sulfate and potassium nitrate. Gunasekaran and Poorniammal [41] reported that the yield, which could be attributed to the need for additional pre-treatment when using this substance to inclusion of peptone in the culture medium led to the highest red pigment yield by Penicillium sp. matchand thevarious growth other requirements pigment-producing of the fungi. fungal strain.

FigureFigure 3. 3.E ffEffectect of of carbon carbon (a(a)) andand nitrogennitrogen ( (bb)) sources sources on on biomass biomass and and total total red red pigment pigment production production by Monascusby Monascus ruber ruberOMNRC45 OMNRC45 in liquidin liquid medium. medium.

ForIn similar this study, reasons, temperature the source and of nitrogenpH were inco thensidered culture important medium environmental is an essential and factor culture influencing microbialparameters growth for improving as well as the the yield production of red dyes of from primary M. ruber and /OMNRC45.or secondary Cultivation bioactive temperature metabolites [40]. Asplays secondary an important metabolites, role thein productionthe rate of ofall both microbial intracellular biochemical and extracellular activities, including pigments nutrient is expected to be influencedabsorption, (quantitativelyenzyme synthesis, and and qualitatively) pigment production by the nitrogen [42]. The source optimum used. temperature We investigated for growth the effects and production of dyes by Monascus spp. has a wide range (from 25 to 37 °C) and primarily depends of organic (peptone–urea) and inorganic (NH NO -(NH ) SO -KNO ) nitrogen sources on the growth on the species [29]. Therefore, an experiment4 was3 conducted4 2 4to determine3 the effect of different incubation temperatures (20–40 °C) on the biomass and red pigment yield from M. ruber OMNRC45. The data obtained and presented in Figure 4a showed that 30 °C was the optimum temperature for maximum biomass and red pigment production. The production of the red pigment increased gradually with a rise in the fermentation temperature from 20 °C to 25 °C and then to 30 °C. Appl. Sci. 2020, 10, 8867 8 of 15 of M. ruber OMNRC45 and extracellular red pigment productivity. As shown in Figure3b, M. ruber OMNRC45 cultured using peptone as the nitrogen source (in cultivation medium) showed the highest pigment yield. However, the increase in fungal biomass production using the same nitrogen source was not significant compared to the results obtained using ammonium sulfate and potassium nitrate. Gunasekaran and Poorniammal [41] reported that the inclusion of peptone in the culture medium led to the highest red pigment yield by Penicillium sp. and various other pigment-producing fungi. In this study, temperature and pH were considered important environmental and culture parameters for improving the yield of red dyes from M. ruber OMNRC45. Cultivation temperature Appl. Sci. 2020, 10, x FOR PEER REVIEW 8 of 15 plays an important role in the rate of all microbial biochemical activities, including nutrient absorption, enzymeSubsequently, synthesis, pigment and pigment production production decreased [42]. as The the optimumincubationtemperature temperature increased for growth from and 30 production °C to of dyes35 °C by andMonascus then to spp.40 °C. has Similar a wide findings range were (from repo 25rted to 37 by◦ JeonC) and et al. primarily [43] on the depends optimum on temperature the species [29]. Therefore,for pigment an experiment production was by M. conducted purpureus to MMK2, determine M. ruber the KCTC6122, effect of di ffanderent M. incubationpurpureus P-57. temperatures More (20–40recently,◦C) on Padmavathi the biomass and and Prabhudessai red pigment [44] yield reported from thatM. optimum ruber OMNRC45. mycelium growth The data and obtainedpigment and presentedproduction in Figure by 4M.a showedanguineus that and 30 M.◦C waspurpureus the optimum MTCC410 temperature were observed for maximumat a fermentation biomass and red pigmenttemperature production. of 30 °C. However, The production these results of the were red slightly pigment different increased from those gradually reported with by Babitha a rise in the et al. [45], who found that the maximum pigment yield was obtained between 32 °C and 35 °C. fermentation temperature from 20 ◦C to 25 ◦C and then to 30 ◦C. Subsequently, pigment production It is well known that the initial pH of the culture medium has a significant effect on enzyme and decreased as the incubation temperature increased from 30 C to 35 C and then to 40 C. Similar findings pigment production by Monascus spp. [46]. Accordingly, ◦an experiment◦ was designed◦ to study the wereeffect reported of different by Jeon pH et values al. [43 ]on on biomass the optimum and red temperature pigment production for pigment by M. productionruber OMNRC45. by M. When purpureus MMK2,the M.fungal ruber isolateKCTC6122, was cultured and M.in a purpureus liquid mediumP-57. un Moreder different recently, initial Padmavathi pH values and (4.5–8.5), Prabhudessai pH 6.5 [44] reportedwas found that optimum to be optimal mycelium for pigment growth production, and pigment as shown production in Figure by 4b.M. Evidently, anguineus anand alkalineM. purpureus pH MTCC410(8.5) strongly were observed inhibited atboth a fermentation biomass and extracellular temperature pigment of 30 ◦ C.produc However,tion. Joshi these et resultsal. [47] reported were slightly differentthat pH from values those ranging reported from by 5.5 Babitha to 6.5 were et al. ideal [45], for who thefound production that theof pigments maximum by pigmentMonascus yieldspp., was while Babitha et al. [48] reported (for M. purpureus) a wider optimal pH range (4.5–7.5) for the same. obtained between 32 ◦C and 35 ◦C.

FigureFigure 4. E ff4.ect Effect of incubationof incubation temperature temperature ((a) and initial initial pH pH (b ()b on) on biomass biomass and and total total red pigment red pigment production by Monascus ruber OMNRC45. production by Monascus ruber OMNRC45.

It3.3. is Biosafety well known Evaluation that of the the initialMonascus pH Pigments of the culture medium has a significant effect on enzyme and pigmentIn addition production to pigments, by Monascus the mycotoxinspp. [46 citrinin]. Accordingly, is also produced an experiment under certain was conditions designed as to a study the ebyproductffect of di offferent fermentation pH values by Monascus on biomass spp. Citrinin and red has pigment hepatotoxic production properties and by M.causes ruber functionalOMNRC45. Whenand the structural fungal isolatedamage was to the cultured kidneys, in in aaddition liquid mediumto altering underliver metabolism different initial[49–51]. pH Several values toxicity (4.5–8.5), pH 6.5studies was foundon Monascus to be optimalpigmentsfor have pigment confirmed production, the safety asof showntheir consumption in Figure4 b.in Evidently,specific quantities. an alkaline Conversely, several studies have confirmed the potential toxicity of some Monascus strains owing to pH (8.5) strongly inhibited both biomass and extracellular pigment production. Joshi et al. [47] reported citrinin contamination, whereas some strains have been reported to be devoid of citrinin production that pH values ranging from 5.5 to 6.5 were ideal for the production of pigments by Monascus spp., ability. In addition to its dependence on the type of fungal strain, citrinin production is also markedly whileinfluenced Babitha etbyal. the [ 48environmental] reported (for and M.culture purpureus conditions) a wider during optimal fungal growth pH range [11,52–54]. (4.5–7.5) In the for present the same. study, we aimed to evaluate the biosafety of the red pigments produced by M. ruber OMNRC45. The 3.3. Biosafety Evaluation of the Monascus Pigments biosafety assessment was performed by citrinin detection (using TLC) in the extracted red pigments in Inparallel addition with the to pigments,measurement the of mycotoxinits toxicity in citrininmice. is also produced under certain conditions as a byproductThe of results fermentation of TLC analyses by Monascus revealedspp. that Citrinin no citrinin has hepatotoxic(nephrotoxic propertiesor hepatotoxic) and was causes detected functional in the chloroform extract or the filtrate of M. ruber OMNRC45 cultured in potato dextrose broth (PDB), PDB plus glycine, or rice media (Figure 5). In TLC, the reference sample (citrinin) formed a yellow fluorescence band under UV light, whereas similar bands were not observed in the target samples. This result is consistent with those reported by Chen et al. [20] and Moharram et al. [28], who confirmed the presence of genes associated with citrinin biosynthesis in only 18 out of 30 Monascus strains studied. Notably, heat treatment of citrinin (140 °C) leads to the production of a novel compound, citrinin H2, which exerts weaker cytotoxic effects than citrinin [55]. Hence, if Appl. Sci. 2020, 10, 8867 9 of 15 and structural damage to the kidneys, in addition to altering liver metabolism [49–51]. Several toxicity studies on Monascus pigments have confirmed the safety of their consumption in specific quantities. Conversely, several studies have confirmed the potential toxicity of some Monascus strains owing to citrinin contamination, whereas some strains have been reported to be devoid of citrinin production ability. In addition to its dependence on the type of fungal strain, citrinin production is also markedly influenced by the environmental and culture conditions during fungal growth [11,52–54]. In the present study, we aimed to evaluate the biosafety of the red pigments produced by M. ruber OMNRC45. The biosafety assessment was performed by citrinin detection (using TLC) in the extracted red pigments in parallel with the measurement of its toxicity in mice. The results of TLC analyses revealed that no citrinin (nephrotoxic or hepatotoxic) was detected in the chloroform extract or the filtrate of M. ruber OMNRC45 cultured in potato dextrose broth (PDB), PDB plus glycine, or rice media (Figure5). In TLC, the reference sample (citrinin) formed a yellow fluorescence band under UV light, whereas similar bands were not observed in the target samples. This result is consistent with those reported by Chen et al. [20] and Moharram et al. [28], who confirmed the presence of genes associated with citrinin biosynthesis in only 18 out of 30 Monascus

Appl.strains Sci. 2020 studied., 10, x FOR Notably, PEER REVIEW heat treatment of citrinin (140 ◦C) leads to the production of a9 novel of 15 compound, citrinin H2, which exerts weaker cytotoxic effects than citrinin [55]. Hence, if present in presentfood materials, in food citrinin-contaminatedmaterials, citrinin-contaminated pigments should pigments be subjected should be to subjecte heat treatmentd to heat or treatment degradation or viadegradation other strategies. via other Based strategies. on the aforementionedBased on the af results,orementioned it can be results, concluded it can that beM. concluded ruber OMNRC45 that M. rubercultured OMNRC45 under the cultured conditions under described the conditions in the describ presented study in the can present be considered study can safebe considered and devoid safe of andcitrinin devoid contamination. of citrinin contamination.

Figure 5. FermentationFermentation of of MonascusMonascus ruber ruber OMNRC45OMNRC45 in inrice rice (a), (a cultural), cultural filtrate filtrate (b), ( band), and biomass biomass (c) (onc) potato on potato dextrose dextrose broth broth (PDB) (PDB) media. media. TLC analysis TLC analysis(d) for citrinin (d) for detection: citrinin lanes detection: 1 and 2—reference lanes 1 and citrinin;2—reference lanes citrinin; 3, 4, and lanes 5—chloroform 3, 4, and 5—chloroform extract of M. extractruber cultivated of M. ruber oncultivated PDB, PDB on plus PDB, glycine, PDB plusand riceglycine, media, and respectively. rice media, respectively.

The proximate composition of red Monascus rice was determined. It It contained contained 9.26% 9.26% moisture, moisture, 72.67% carbohydrate, carbohydrate, 11.89% 11.89% protein, protein, 1.34% 1.34% lipid, lipid, and and 0.42% 0.42% ash. ash. These These values values were weresimilar similar to those to reportedthose reported by Kumari by Kumari et al. [31]. et al.The [31 biotox]. Theicity biotoxicity of the red ofpigments the red obtained pigments from obtained M. ruber from OMNRC45M. ruber wasOMNRC45 evaluated was in evaluated a mouse feeding in a mouse experiment. feeding experiment.Mice were fed Mice with were fungal fed pigments with fungal at oral pigments doses of at 0,oral 10, doses 100, of1000, 0, 10, 1600, 100, 1000,2900, 1600, and 2900,5000 andmg/kg 5000 body mg/ kgweight body weightin distilled in distilled water. water. The artificial The artificial dye carmoisinedye carmoisine E122 E122 was wasused used as a aspositive a positive control control for comparison. for comparison. At the At end the endof the of experiment, the experiment, the followingthe following organs organs were were harvested: harvested: the the heart, heart, liver, liver, kidney, kidney, and and spleen. spleen. Changes Changes in in organ organ appearance appearance were examined, and the organs were weighed. The hepatosomatic index (HIS), mortality percentage, and toxicity signs (abnormal signs) were evaluated (Table 1). Body and organ weights and the ratio of liver to body weight are indicators of organ damage or abnormalities resulting from the provision of treatment [56]. The results showed that there were significant differences between the control and Monascus and carmoisine pigment-fed mice at different concentrations in terms of liver, kidney, heart, and spleen weight. In addition, visual examination of the organs after dissection showed that organs harvested from mice treated with the highest concentrations of Monascus pigment showed normal signs, similar to the organs harvested from control mice. However, this was not the case for the mice fed carmoisine, especially at 5000 mg/kg. At this concentration, marginal changes were observed in liver color and size. Moreover, no mortality was observed in mice fed the Monascus pigment. According to Masango et al. [57], mortality with characteristic enlarged liver (liver weight/body mass >7%) is used to measure hepatotoxicity. There was no hepatotoxicity observed in mice treated with different concentrations of the pigment, since the ratio ranged between 5.4 and 5.7; however, the HIS value increased, especially in mice treated with carmoisine at different concentrations. Further, aggressive cases were observed in male mice treated with 5000 mg/kg of carmoisine, while cases of diarrhea and deaths were observed in both females and males. The LD50 value of carmoisine (determined via a simple toxicity assessment) was found to be 4166 mg/kg body weight.

Appl. Sci. 2020, 10, 8867 10 of 15 were examined, and the organs were weighed. The hepatosomatic index (HIS), mortality percentage, and toxicity signs (abnormal signs) were evaluated (Table1). Body and organ weights and the ratio of liver to body weight are indicators of organ damage or abnormalities resulting from the provision of treatment [56]. The results showed that there were significant differences between the control and Monascus and carmoisine pigment-fed mice at different concentrations in terms of liver, kidney, heart, and spleen weight. In addition, visual examination of the organs after dissection showed that organs harvested from mice treated with the highest concentrations of Monascus pigment showed normal signs, similar to the organs harvested from control mice. However, this was not the case for the mice fed carmoisine, especially at 5000 mg/kg. At this concentration, marginal changes were observed in liver color and size. Moreover, no mortality was observed in mice fed the Monascus pigment. According to Masango et al. [57], mortality with characteristic enlarged liver (liver weight/body mass >7%) is used to measure hepatotoxicity. There was no hepatotoxicity observed in mice treated with different concentrations of the pigment, since the ratio ranged between 5.4 and 5.7; however, the HIS value increased, especially in mice treated with carmoisine at different concentrations. Further, aggressive cases were observed in male mice treated with 5000 mg/kg of carmoisine, while cases of diarrhea and deaths were observed in both females and males. The LD50 value of carmoisine (determined via a simple toxicity assessment) was found to be 4166 mg/kg body weight.

Table 1. Effect of Monascus pigment and carmoisine E122 at different concentrations on mean organ weight (g), hepatotoxicity, mortality, and other observations.

Concentration Body Weight Liver Kidney Heart Spleen L/B% H M 1 (mg/kg bw Mice) Control 26.04 0.52 1.42 0.12 0.16 0.14 0.14 0.10 0.12 0.15 5.46 No ± ± ± ± ± 10 M 27.26 0.58 1.50 0.25 0.17 0.11 0.15 0.09 0.12 0.08 5.51 No ± ± ± ± ± 10 C 26.11 0.41 1.46 0.02 0.16 0.04 0.14 0.02 0.12 0.18 5.60 No ± ± ± ± ± 100 M 26.50 0.23 1.48 0.05 0.16 0.21 0.14 0.10 0.12 0.15 5.60 No ± ± ± ± ± 100 C 27.80 0.44 1.56 0.05 0.17 0.01 0.15 0.07 0.13 0.08 5.60 No ± ± ± ± ± 1000 M 28.40 0.15 1.59 0.03 0.17 0.09 0.15 0.04 0.13 0.06 5.60 No ± ± ± ± ± 1000 C 27.28 0.54 1.55 0.01 0.18 0.04 0.15 0.06 0.13 0.08 5.70 No ± ± ± ± ± 1600 M 29.23 0.24 1.66 0.03 0.18 0.05 0.15 0.04 0.13 0.06 5.70 No ± ± ± ± ± 1600 C 28.11 0.29 1.71 0.04 0.19 0.07 0.16 0.09 0.13 0.30 6.10 No ± ± ± ± ± 2900 M 29.73 0.88 1.70 0.16 0.18 0.03 0.16 0.05 0.14 0.06 5.73 No ± ± ± ± ± 2900 C 28.61 0.98 1.83 0.06 0.207 0.04 0.16 0.06 0.14 0.02 6.40 No ± ± ± ± ± 5000 M 29.73 0.65 1.71 0.09 0.18 0.07 0.16 0.04 0.15 0.03 5.73 No ± ± ± ± ± 5000 C 28.61 0.98 1.95 0.05 0.20 0.02 0.17 0.03 0.14 0.02 6.80 30% ± ± ± ± ± M, Monascus red pigment; C, carmoisine pigment; M 1, mortality; H, hepatotoxicity: mortality with characteristic enlarged liver (liver weight/body mass (L/B) % >7) was used as the measure of hepatotoxicity.

Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) activities are indicators of hepatic health. Our study demonstrated that the Monascus pigment exerted no significant effect on AST, ALT, and ALP levels compared to the control group (Table2). Blood urea nitrogen (BUN) is an indicator of kidney damage resulting from the enzymatic hydrolysis of urea to ammonia by urease [58]. The current study showed that there were no differences in BUN levels in mice fed the Monascus pigment compared to the levels in control mice. The results for blood enzyme levels in mice fed the red Monascus pigments from M. ruber OMNRC45 were similar to those obtained by Kumari et al. [31]. However, the carmoisine-fed mice showed significant differences in the levels of AST, ALT, and ALP compared to the Monascus pigment-fed and control mice, possibly owing to the effect of the synthetic dye. Although the values of most parameters evaluated in our study were within the normal range, similar to findings reported by Serfilippi et al. [59], the values increased significantly at high concentrations of carmoisine. Appl. Sci. 2020, 10, 8867 11 of 15

Table 2. Effects of Monascus pigment and carmoisine E122 at different concentrations on the biological

activitiesAppl. Sci. of2020 mice., 10, x FOR PEER REVIEW 11 of 15 Appl. Sci. 2020, 10, x FOR PEER REVIEW 11 of 15 Concentration AST U/L ALT U/L ALP U/L BUN mg/dL Table(mg/ 2.kg Effects bw mice) of Monascus pigment and carmoisine E122 at different concentrations on the biological activitiesTable 2.Control Effects of mice. of Monascus 76.02 pigment0.06 and carmoisine 74.15 0.07 E122 at 38.56 differe0.24nt concentrations 16.39 0.12 on the biological activities of mice. ± ± ± ± ConcentrationM 76.03 0.10 74.11 0.02 38.61 0.05 16.34 0.12 10 ±AST U/L ± ALT U/L ± ALP U/L ± BUN mg/dL (mg/kgConcentration bw mice)C 77.36 1.31 77.11 1.59 39.60 4.51 17.67 1.22 ±AST U/L ± ALT U/L ± ALP U/L ± BUN mg/dL M 76.08 0.07 74.17 0.12 38.53 0.26 16.40 0.17 (mg/kg100 Control bw mice) 76.02± ± 0.06 ± 74.15 ± 0.07 ± 38.56 ± 0.24± 16.39 ± 0.12 C 77.75 0.82 77.84 0.81 40.12 0.44 18.07 0.72 Control M 76.0376.02± ± 0.100.06 ± 74.1174.15 ± 0.020.07 ± 38.6138.56 ± 0.050.24± 16.3416.39 ± 0.12 10 M 76.10 0.06 74.14 0.04 38.73 0.08 16.33 0.22 100 MC 77.3676.03± ± 1.310.10 ± 77.1174.11 ± 1.590.02 ± 39.60 38.61 ± 4.510.05± 17.6716.34 ± 1.220.12 10 C 78.10 0.95 78.81 1.42 42.06 0.20 19.66 0.26 MC 76.0877.36± ± 0.071.31 ± 74.1777.11 ± 0.121.59 ± 38.5339.60 ± 0.264.51± 16.4017.67 ± 0.171.22 100 M 76.04 0.12 74.19 0.06 38.46 0.12 16.30 0.10 1600 MC 77.7576.08± ± 0.820.07 ± 77.8474.17 ± 0.810.12 ± 40.12 38.53 ± 0.440.26± 18.0716.40 ± 0.720.17 100 C 78.38 1.26 79.86 2.28 43.10 2.83 20.30 0.48 MC 76.1077.75± ± 0.060.82 ± 74.1477.84 ± 0.040.81 ± 38.7340.12 ± 0.080.44± 16.3318.07 ± 0.220.72 100 M 76.09 0.17 74.18 0.06 38.59 0.09 16.45 0.28 2900 MC 78.1076.10± ± 0.950.06 ± 78.8174.14 ± 1.420.04 ± 42.06 38.73 ± 0.200.08± 19.6616.33 ± 0.260.22 100 C 81.09 1.69 80.85 3.34 46.59 1.43 23.11 0.10 MC 76.0478.10± ± 0.120.95 ± 74.1978.81 ± 0.061.42 ± 38.4642.06 ± 0.120.20± 16.3019.66 ± 0.100.26 1600 M 76.18 0.11 74.19 0.08 38.57 0.23 16.39 0.13 5000 MC 78.3876.04± ± 1.260.12 ± 79.8674.19 ± 2.280.06 ± 43.10 38.46 ± 2.830.12± 20.3016.30 ± 0.480.10 1600 C 92.85 2.12 92.86 1.85 53.91 2.67 28.39 0.68 MC 76.0978.38± ± 0.171.26 ± 74.1879.86 ± 0.062.28 ± 38.5943.10 ± 0.092.83± 16.4520.30 ± 0.280.48 M, Monascus2900 pigment; C, carmoisine dye; AST, aspartate aminotransferase; ALT, alanine aminotransferase; MC 81.0976.09 ± 1.690.17 80.8574.18 ± 3.340.06 46.59 38.59 ± 1.430.09 23.1116.45 ± 0.100.28 ALP, alkaline2900 phosphatase; BUN, blood urea nitrogen; dL, deciliter. MC 76.1881.09 ± 0.111.69 74.1980.85 ± 0.083.34 38.5746.59 ± 0.231.43 16.3923.11 ± 0.130.10 5000 MC 92.8576.18 ± 2.120.11 92.8674.19 ± 1.850.08 53.91 38.57 ± 2.670.23 28.3916.39 ± 0.680.13 Based5000 on the results of the biosafety evaluation and findings reported by Serfilippi et al. [59], M, Monascus pigment;C C, carmoisine 92.85 dye;± 2.12 AST, aspartate 92.86 aminotransferase; ± 1.85 53.91 ALT, ± alanine 2.67 aminotransferase; 28.39 ± 0.68 the natural red pigment obtained from M. ruber OMNRC45 can be considered a promising natural ALP,M, Monascus alkaline pigment; phosphatase; C, carmoisine BUN, blood dye; urea AST, nitrogen; aspartate dL, aminotransferase; deciliter. ALT, alanine aminotransferase; colorant. Conversely, the application of the synthetic dye carmoisine can induce several abnormal ALP, alkaline phosphatase; BUN, blood urea nitrogen; dL, deciliter. signs in mice, especially at high concentrations, and it must be replaced with a safe natural product. 3.4. Sensory Evaluation of Food Products 3.4. Sensory Evaluation of Food Products 3.4. SensoryTwo Evaluation food products of Food Products were evaluated as models for food industries, and the results of sensory Two food products were evaluated as models for food industries, and the results of sensory Twoscores food are products presented were in Table evaluated 3. The asfood models products for food(lollipops industries, and jellybeans and the) resultsacquired of an sensory intense and scores are presented in Table 3. The food products (lollipops and jellybeans) acquired an intense and scorespersistent are presented red color, in Table and3. Thetheir foodorganoleptic products properties (lollipops andwere jellybeans) enhanced acquiredupon the an addition intense of red persistent red color, and their organoleptic properties were enhanced upon the addition of red and persistentpigments red from color, M. andruber their OMNRC45. organoleptic The appeal, properties color, were appearance, enhanced and upon overall the addition acceptability of red of the pigments from M. ruber OMNRC45. The appeal, color, appearance, and overall acceptability of the pigmentslollipops from M.and ruber jellybeansOMNRC45. were considered The appeal, to color, have appearance,improved owing and overall to the acceptabilityuse of the natural of the fungal lollipops and jellybeans were considered to have improved owing to the use of the natural fungal lollipopscolorant. and jellybeans As stated werein previous considered studies, to have the application improved owingof natural to the dyes use can of thebe potentially natural fungal beneficial colorant. As stated in previous studies, the application of natural dyes can be potentially beneficial colorant.for Asprotecting stated in consumer previous studies, health thebecause application it allows ofnatural the manufacturing dyes can be potentially of completely beneficial natural for foods for protecting consumer health because it allows the manufacturing of completely natural foods protectingdevoid consumer of artificial health additives because [60,61]. it allows the manufacturing of completely natural foods devoid of devoid of artificial additives [60,61]. artificial additives [60,61]. Table 3. Mean sensory scores of food samples colored with the red pigment from Monascus ruber Table 3.OMNRC45.TableMean 3. Mean sensory sensory scores scores of food of food samples samples colored colored with with the the red red pigment pigment from fromMonascus Monascus ruber OMNRC45. ruberFoodOMNRC45. Sample of Overall Color Taste Texture Flavor ProductFood SampleProduct of AcceptabilityOverall Food Sample of Color Taste Texture Flavor Overall Product Product Color Taste Texture Flavor Acceptability Product Product Acceptability

Lollipops 9.30 ± 0.15 9.09 ± 0.30 8.97 ± 0.20 9.50 ± 0.21 9.21 ± 0.07 LollipopsLollipops 9.309.30 ± 0.15 9.09 9.09 0.30± 0.30 8.97 8.970.20 ± 0.20 9.50 9.500.21 ± 0.21 9.21 0.07 9.21 ± 0.07 ± ± ± ± ±

JellybeansJellybeans 9.389.38 ± 0.08 9.03 9.03 0.19± 0.19 9.30 9.300.26 ± 0.26 9.60 9.600.15 ± 0.15 9.33 0.06 9.33 ± 0.06 Jellybeans 9.38 ±± 0.08 9.03± ± 0.19 9.30± ± 0.26 ± 9.60 ± 0.15 ± 9.33 ± 0.06

4. Conclusions 4. Conclusions The fungal strain M. ruber OMNRC45 was shown to effectively produce a natural red pigment underThe submerged fungal strain fermentation M. ruber OMNRC45 conditions waswithout shown producing to effectively the harmful produce mycotoxin a natural redcitrinin. pigment The productionunder submerged of fungal fermentation pigments could conditions be maximized without producingby controlling the harmfulfermentation mycotoxin conditions citrinin. such The as carbonproduction and nitrogenof fungal sources pigments and could the pH be value.maximized Sensory by andcontrolling biosafety fermentation evaluation of conditions the red Monascus such as pigmentcarbon and produced nitrogen by sources M. ruber and OMNRC45 the pH value. indicated Sensory its biosafetyand biosafety and applicabilityevaluation of as the a foodred Monascus colorant pigment produced by M. ruber OMNRC45 indicated its biosafety and applicability as a food colorant Appl. Sci. 2020, 10, 8867 12 of 15

4. Conclusions The fungal strain M. ruber OMNRC45 was shown to effectively produce a natural red pigment under submerged fermentation conditions without producing the harmful mycotoxin citrinin. The production of fungal pigments could be maximized by controlling fermentation conditions such as carbon and nitrogen sources and the pH value. Sensory and biosafety evaluation of the red Monascus pigment produced by M. ruber OMNRC45 indicated its biosafety and applicability as a food colorant compared to unsafe artificial colorants. Further studies should be conducted to better understand the sources, production, and biosafety of microbial pigments as promising alternatives to hazardous artificial colorants.

Author Contributions: All authors conceived and planned the study; O.M.D. and I.A.M. performed the microbiological experiments; H.S.A. and S.A.A. performed the sensory and biosafety evaluation. All authors contributed equally to data analysis and discussion of results and commented on the manuscript. O.M.D. and I.A.M. wrote the manuscript in consultation with all authors; I.A.M. reviewed and edited the manuscript; Y.-K.O. revised and consolidated the final version of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The research team expresses their gratefulness to King Saud University (project number: RSP-2020/283), Riyadh, Saudi Arabia, and the National Research Centre, Cairo, Egypt, for the financial support of this work. Y.-K. Oh would also like to acknowledge the support of the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT, Republic of Korea (NRF-2019R1A2C1003463). Conflicts of Interest: The authors declare no conflict of interest.

References

1. Barakat, K.M.; Mattar, M.Z.; Sabae, S.Z.; Darwesh, O.M.; Hassan, S.H. Production and characterization of bioactive pyocyanin pigment by marine Pseudomonas aeruginosa OSh1. Res. J. Pharm. Biol. Chem. Sci. 2015, 6, 933–943. 2. Darwesh, O.M.; Barakat, K.M.; Mattar, M.Z.; Sabae, S.Z.; Hassan, S.H. Production of antimicrobial blue green pigment Pyocyanin by marine Pseudomonas aeruginosa. Biointerface Res. Appl. Chem. 2019, 9, 4334–4339. 3. Wibowo, S.; Vervoort, L.; Tomic, J.; Santiago, J.S.; Lemmens, L.; Panozzo, A.; Grauwet, T.; Hendrickx, M.; Van Loey, A. Colour and carotenoid changes of pasteurised orange juice during storage. Food Chem. 2015, 171, 330–340. [CrossRef][PubMed] 4. Ogbodo, U.O.; Ugwuanyi, J.O. Production, Use, and Prospects of Microbial Food Colorants. In Microbial Production of Food Ingredients and Additives; Elsevier Inc.: Academic Press: London, UK, 2017; Volume 2017, pp. 189–216. 5. Nigam, P.S.; Luke, J.S. Food additives: Production of microbial pigments and their antioxidant properties. Curr. Opin. Food Sci. 2016, 7, 93–100. [CrossRef] 6. Vendruscolo, F.; Bühler, R.M.M.; de Carvalho, J.C.; de Oliveira, D.; Moritz, D.E.; Schmidell, W.; Ninow, J.L. Monascus: A reality on the production and application of microbial pigments. Appl. Biochem. Biotechnol. 2016, 178, 211–223. [CrossRef] 7. Malik, K.; Tokkas, J.; Goyal, S. Microbial pigments: A review. Int. J. Microbial. Res. Technol. 2012, 1, 361–365. 8. Kim, Y.E.; Matter, I.A.; Lee, N.; Jung, M.; Lee, Y.C.; Choi, S.A.; Lee, S.Y.; Kim, J.R.; Oh, Y.K. Enhancement of astaxanthin production by Haematococcus pluvialis using magnesium aminoclay nanoparticles. Bioresour. Technol. 2020, 307, 123270. [CrossRef] 9. Jongrungruangchok, S.; Kittakoop, P.; Yongsmith, B.; Bavovada, R.; Tanasupawat, S.; Lartpornmatulee, N.; Thebtaranonth, Y. Azaphilone pigments from a yellow mutant of the fungus . Phytochemistry 2004, 65, 2569–2575. [CrossRef] 10. Patakova, P. Monascus secondary metabolites: Production and biological activity. J. Ind. Microbiol. Biotechnol. 2013, 40, 169–181. [CrossRef] 11. Feng, Y.; Chen, W.; Chen, F.A. Monascus pilosus MS-1 strain with high-yield monacolin K but no citrinin. Food Sci. Biotechnol. 2016, 25, 1115–1122. [CrossRef] Appl. Sci. 2020, 10, 8867 13 of 15

12. Mostafa, M.E.; Abbady, M.S. Secondary metabolites and bioactivity of the Monascus pigments review article. Glob. J. Biotechnol. Biochem. 2014, 9, 1–13. 13. Yu, X.; Wu, H.; Zhang, J. Effect of Monascus as a nitrite substitute on color, lipid oxidation, and proteolysis of fermented meat mince. Food Sci. Biotechnol. 2015, 24, 575–581. [CrossRef] 14. Rosales, E.; Pazos, M.; Sanromán, M.Á. Solid-state fermentation for food applications. In Current Developments in Biotechnology and Bioengineering; Elsevier: Amsterdam, The Netherlands, 2018; Volume 2018, pp. 319–355. 15. Mondal, S.; Pandit, S.G.; Puttananjaiah, M.H.; Harohally, N.V.; Dhale, M.A. Structural and functional characterization of new pigment molecule Monashin from Monascus purpureus CFR410–11. Process Biochem. 2019, 82, 173–178. [CrossRef] 16. Wu, W.T.; Wang, P.M.; Chang, Y.Y.; Huang, T.K.; Chien, Y.H. Suspended rice particles for cultivation of Monascus purpureus in a tower-type bioreactor. Appl. Microbiol. Biotechnol. 2000, 53, 542–544. [CrossRef] [PubMed] 17. Domínguez-Espinosa, R.M.; Webb, C. Submerged fermentation in wheat substrates for production of Monascus pigments. World J. Microbiol. Biotechnol. 2003, 19, 329–336. [CrossRef] 18. Darwesh, O.M.; El-Maraghy, S.H.; Abdel-Rahman, H.M.; Zaghloul, R.A. Improvement of paper wastes conversion to bioethanol using novel cellulose degrading fungal isolate. Fuel 2020, 262, 116518. [CrossRef] 19. Hsu, F.L.; Wang, P.M.; Lu, S.Y.; Wu, W.T. A combined solid-state and submerged cultivation integrated with adsorptive product extraction for production of Monascus red pigments. Bioprocess. Biosyst. Eng. 2002, 25, 165–168. 20. Chen, Y.P.; Tseng, C.P.; Chien, I.L.; Wang, W.Y.; Liaw, L.L.; Yuan, G.F. Exploring the distribution of citrinin biosynthesis related genes among Monascus species. J. Agric. Food. Chem. 2008, 56, 11767–11772. [CrossRef] 21. Lin, Y.L.; Wang, T.H.; Lee, M.H.; Su, N.W. Biologically active components and nutraceuticals in the Monascus-fermented rice: A review. Appl. Microbiol. Biotechnol. 2008, 77, 965–973. [CrossRef] 22. Iriart, X.; Fior, A.; Blanchet, D.; Berry, A.; Neron, P.; Aznar, C. Monascus ruber: Invasive gastric infection caused by dried and salted fish consumption. J. Clin. Microbiol. 2010, 48, 3800–3802. [CrossRef] 23. Darwesh, O.M.; Eida, M.F.; Matter, I.A. Isolation, screening and optimization of L-asparaginase producing bacterial strains inhabiting agricultural soils. Biosci. Res. 2018, 15, 2802–2812. 24. Elshahawy, I.; Abouelnasr, H.M.; Lashin, S.M.; Darwesh, O.M. First report of Pythium aphanidermatum infecting tomato in Egypt and its control using biogenic silver nanoparticles. J. Plant Prot. Res. 2018, 58, 137–151. 25. Hasanin, M.S.; Darwesh, O.M.; Matter, I.A.; El-Saied, H. Isolation and characterization of non-cellulolytic Aspergillus flavus EGYPTA5 exhibiting selective ligninolytic potential. Biocatal. Agric. Biotechnol. 2019, 17, 160–167. [CrossRef] 26. Darwesh, O.M.; El-Hawary, A.S.; El Kelany, U.S.; El-Sherbiny, G.M. Nematicidal activity of thermostable alkaline protease produced by Saccharomonospora viridis strain Hw G550. Biotechnol. Rep. 2019, 24, e00386. [CrossRef][PubMed] 27. Orozco, S.F.B.; Kilikian, B.V. Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802. World J. Microbiol. Biotechnol. 2008, 24, 263–268. [CrossRef] 28. Moharram, A.M.; Mostafa, M.E.; Ismail, M.A. Chemical profile of Monascus ruber strains. Food Technol. Biotechnol. 2012, 50, 490–499. 29. Carvalho, J.C.; Oishi, B.O.; Pandey, A.; Soccol, C.R. Biopigments from Monascus: Strains selection, citrinin production and color stability. Braz. Arch. Biol. Technol. 2005, 48, 885–894. [CrossRef] 30. Elsayed, M.E.; Salem, M.A.; Selem, S.B. Effect of feeding at different levels of chromium picolinate and magnesium sulfate on diabetic rats. J. Food Dairy Sci. 2015, 6, 379–392. [CrossRef] 31. Kumari, H.M.; Naidu, K.A.; Vishwanatha, S.; Narasimhamurthy, K.; Vijayalakshmi, G. Safety evaluation of Monascus purpureus red mould rice in albino rats. Food Chem. Toxicol. 2009, 47, 1739–1746. [CrossRef] 32. Akilandeswari, P.; Pradeep, B.V. Exploration of industrially important pigments from soil fungi. Appl. Microbiol. Biotechnol. 2016, 100, 1631–1643. [CrossRef] 33. Bouksir, K.; Kazzaz, M.; Fehri, H.F.; Bouziane, H.; Bouksir, H.; El Haskouri, F. Monascus ruber: A new of onychomycosis in the north of Morocco (Tetouan). J. Mycol. Med. 2018, 28, 502–509. [CrossRef][PubMed] 34. Eida, M.F.; Darwesh, O.M.; Matter, I.A. Cultivation of oleaginous microalgae Scenedesmus obliquus on secondary treated municipal wastewater as growth medium for biodiesel production. J. Ecol. Eng. 2018, 19, 38–51. [CrossRef] 35. Sadek, Z.I.; Abdel-Rahman, M.A.; Azab, M.S.; Darwesh, O.M.; Hassan, M.S. Microbiological evaluation of infant foods quality and molecular detection of Bacillus cereus toxins relating genes. Toxicol. Rep. 2018, 5, 871–877. [CrossRef][PubMed] Appl. Sci. 2020, 10, 8867 14 of 15

36. Kheiralla, Z.H.; Hewedy, M.A.H.; Mohammed, H.R.; Darwesh, O.M. Isolation of pigment producing actinomycetes from rhizosphere soil and application it in textiles dyeing. Res. J. Pharm. Biol. Chem. Sci. 2016, 7, 2128–2136. 37. Chatterjee, S.; Maity, S.; Chattopadhyay, P.; Sarkar, A.; Laskar, S.; Sen, S.K. Characterization of red pigment from Monascus in submerged culture red pigment from Monascus purpureus. J. Appl. Sci. Res. 2008, 5, 2102–2108. 38. Long, C.; Cui, J.; Xie, S.; Zhang, D.; Liu, M.; Zhang, Z.; Huang, Z.; Zeng, B. The alpha-amylase MrAMY1 is better than MrAMY2 in rice starch degradation, which promotes Monascus pigments production in Monascus ruber. 3 Biotech 2020, 10, 1–6. [CrossRef][PubMed] 39. Hilares, R.T.; de Souza, R.A.; Marcelino, P.F.; da Silva, S.S.; Dragone, G.; Mussatto, S.I.; Santos, J.C. Sugarcane bagasse hydrolysate as a potential feedstock for red pigment production by Monascus ruber. Food Chem. 2018, 245, 786–791. [CrossRef] 40. Darwesh, O.M.; Matter, I.A.; Eida, M.F.; Moawad, H.; Oh, Y.K. Influence of nitrogen source and growth phase on extracellular biosynthesis of silver nanoparticles using cultural filtrates of Scenedesmus obliquus. Appl. Sci. 2019, 9, 1465. [CrossRef] 41. Gunasekaran, S.; Poorniammal, R. Optimization of fermentation conditions for red pigment production from Penicillium sp. under submerged cultivation. Afr. J. Biotechnol. 2008, 7, 1894–1898. [CrossRef] 42. Shuler, M.L.; Kargi, F. Bioprocess considerations in using plant cell cultures. In Bioprocess Engineering (Basic Concepts), 2nd ed.; Prentice-Hall Inc.: Englewood Cliffs, NJ, USA, 2002; pp. 405–419. 43. Jeon, C.P.; Lee, J.B.; Choi, S.Y.; Shin, J.W.; Lee, O.S.; Choi, C.S.; Rhee, C.H.; Kwon, G.S. Optimal culture condition for production of water-soluble red pigments by Monascus purpureus. J. Korean Soc. Food Sci. Nutr. 2006, 35, 493–498. 44. Padmavathi, T.; Prabhudessai, T. A solid liquid state culture method to stimulate Monascus pigments by intervention of different substrates. Int. Res. J. Biol. Sci. 2013, 2, 22–29. 45. Babitha, S.; Soccol, C.R.; Pandey, A. Solid-state fermentation for the production of Monascus pigments from jackfruit seed. Bioresour. Technol. 2007, 98, 1554–1560. [CrossRef] 46. Ouyang, W.; Liu, X.; Wang, Y.; Huang, Z.; Li, X. Addition of genistein to the fermentation process reduces citrinin production by Monascus via changes at the transcription level. Food Chem. 2020, 128410. [CrossRef] 47. Joshi, V.K.; Attri, D.; Bala, A.; Bhushan, S. Microbial pigments. Indian J. Biotechnol. 2003, 2, 362–369. 48. Babitha, S.; Soccol, C.R.; Pandey, A. Effect of stress on growth, pigment production and morphology of Monascus sp. in solid cultures. J. Basic Microbiol. 2007, 47, 118–126. [CrossRef][PubMed] 49. Lockard, V.G.; Phillips, R.D.; Hayes, A.W.; Berndt, W.O.; O’Neal, R.M. Citrinin nephrotoxicity in rats: A light and electron microscopic study. Exp. Mol. Pathol. 1980, 32, 226–240. [CrossRef] 50. Kogika, M.M.; Hagiwara, M.K.; Mirandola, R.M. Experimental citrinin nephrotoxicosis in dogs: Renal function evaluation. Vet. Hum. Toxicol. 1993, 35, 136–140. 51. Da Lozzo, E.J.; Oliveira, M.B.M.; Carnieri, E.G.S. Citrinin-induced mitochondrial permeability transition. J. Biochem. Mol. Toxicol. 1998, 12, 291–297. [CrossRef] 52. Lin, T.F.; Demain, A.L. Effect of nutrition of Monascus sp. on formation of red pigments. Appl. Microbiol. Biotechnol. 1991, 36, 70–75. [CrossRef] 53. Blanc, P.; Laussac, J.P.; Le Bars, J.; Le Bars, P.; Loret, M.O.; Pareilleux, A.; Prome, D.; Prome, J.C.; Santerre, A.L.; Goma, G. Characterization of monascidin A from Monascus as citrinin. Int. J. Food Microbiol. 1995, 27, 201–213. [CrossRef] 54. Blanc, P.J.; Loret, M.O.; Goma, G. Production of citrinin by various species of Monascus. Biotechnol. Lett. 1995, 17, 291–294. [CrossRef] 55. Agboyibor, C.; Kong, W.B.; Chen, D.; Zhang, A.M.; Niu, S.Q. Monascus pigments production, composition, bioactivity and its application: A review. Biocatal. Agric. Biotechnol. 2018, 16, 433–447. [CrossRef] 56. Poorniammal, R.; Gunasekaran, S.; Ariharasivakumar, G. Toxicity evaluation of fungal food colourant from Thermomyces sp. in albino mice. J. Sci. Ind. Res. 2011, 70, 773–777. 57. Masango, M.; Myburgh, J.; Botha, C.; Labuschagne, L.; Naicker, D. A comparison of in vivo and in vitro assays to assess the toxicity of algal blooms. Water Res. 2008, 42, 3241–3248. [CrossRef][PubMed] 58. Ammar, A.; Mahmood, H.Z.A.; Shahid, Z.; Jain, R.; Chen, G. Etanercept-associated Nephropathy. Cureus 2019, 11, e5419. [CrossRef][PubMed] 59. Serfilippi, L.M.; Stackhouse Pallman, D.R.; Gruebbel, M.M.; Kern, T.J.; Spainhour, C.B. Assessment of retinal degeneration in outbred albino mice. Comp. Med. 2004, 54, 69–76. [PubMed] Appl. Sci. 2020, 10, 8867 15 of 15

60. Baranova, M.Á.R.I.A.; Mala, P.; Burdova, O.L’.G.A.; Hadbavny, M.; Sabolová, G. Effect of natural pigment of Monascus purpureus on the organoleptic characters of processed cheeses. Bull. Vet. Inst. Pulawy 2004, 48, 59–62. 61. Vidyalakshmi, R.; Paranthaman, R.; Murugesh, S.; Singaravadivel, K. Microbial bioconversion of rice broken to food grade pigments. Glob. J. Biotechnol. Biochem. 2009, 4, 84–87.

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